Stem Cells Lecture Review

Stem Cells: Fundamental Concepts and Applications

Learning Objectives

Explain asymmetric division in stem cells: Understand how a stem cell divides to produce both a replica of itself and a daughter cell destined for differentiation.
Distinguish between different potency levels of stem cells: Differentiate between totipotent, pluripotent, multipotent, and unipotent stem cells.
Describe mechanisms promoting pluripotency in Inner Cell Mass (ICM) cells & key transcription factors: Identify the molecular pathways and specific proteins (transcription factors) essential for maintaining the undifferentiated state of ICM cells.
Explain how the ICM and trophectoderm are formed: Detail the cellular processes leading to the segregation of these two crucial early embryonic structures.
Identify stem cell niches for several adult stem cell types: Locate the specific microenvironments where adult stem cells reside and are regulated.
Understand signaling pathways regulating proliferation and differentiation of neural and hematopoietic stem cells: Explain the major molecular communication networks controlling the behavior of these specific adult stem cells.
Recognize the unique properties of mesenchymal stem cells (MSCs): Highlight the distinct characteristics and broad differentiation potential of MSCs.
Propose a strategy to treat a human disease using induced pluripotent stem (iPS) cells: Outline a theoretical approach for therapeutic intervention using iPS cell technology.

Key Terminology

Asymmetric division: A type of cell division where daughter cells receive different complements of cellular components, leading to different developmental fates.
Self-renewal: The ability of a stem cell to divide and produce an identical daughter stem cell.
Totipotent: Capable of producing all cell types of an embryo and extraembryonic tissues (e.g., placenta).
Pluripotent: Capable of producing all cell types of the embryo proper (but not extraembryonic tissues).
Multipotent: Capable of producing cell types specific for a given tissue or lineage.
Unipotent: Capable of generating only one specific cell type.
Progenitor: A cell that can proliferate through multiple rounds of division and is committed to becoming a particular type of differentiated cell, but has a transient life and limited self-renewal capacity compared to a stem cell.
Inner Cell Mass (ICM): The cluster of cells inside the blastocyst that will develop into the embryo.
Trophectoderm (trophoblast): The outer layer of cells of the blastocyst that gives rise to the placenta and other extraembryonic tissues.
Stem cell niche: The specific microenvironment that surrounds and regulates a stem cell, influencing its self-renewal, survival, and differentiation.
Subgranular zone (SGZ) of the hippocampus: One of the two principal regions in the adult mammalian brain where neural stem cells are found.
Ventricular-subventricular zone (V-SVZ) of the lateral ventricle: The other principal region for neural stem cells in the adult mammalian brain.
B cell (neural stem cell subtype): Specific type of neural stem cell found in the V-SVZ, existing in quiescent and actively proliferating states.
Oscillations of Notch activity: Temporal fluctuations in the activity of the Notch signaling pathway, which regulate stem cell fate.
Hematopoiesis: The process of blood cell formation.
Endosteal niche: A subniche for hematopoietic stem cells (HSCs) located near the bone surface, promoting quiescence.
Perivascular niche: A subniche for HSCs associated with blood vessels, promoting active proliferation and migration.
Long-term and short-term HSCs: Subtypes of HSCs differing in their self-renewal capacity and proliferative activity.
Mesenchymal stem cells (MSCs): Multipotent adult stem cells found in various tissues, playing dual roles as stromal support and stem cells.
Organoid: A rudimentary, 3-dimensional organ-like structure cultured in vitro from stem cells, mimicking embryonic organogenesis.
Induced pluripotent stem cells (iPS cells): Somatic cells that have been genetically reprogrammed to an embryonic stem cell-like pluripotent state.

The Stem Cell Concept

Definition: Undifferentiated cells that possess two key properties:
1. Self-renewal: The ability to divide (via mitosis) to produce more stem cells, essentially a replica of themselves.
2. Potency: The power to differentiate into specialized cell types. A daughter cell from a stem cell division can undergo further development and specialization.
Asymmetric Division: A stem cell can divide asymmetrically, producing one stem cell and one daughter cell committed to differentiation. This mechanism helps stabilize the stem cell pool while generating differentiated progeny.
- Some stem cells may be more prone to symmetric division to maintain the stem cell pool, while others are geared towards producing differentiated cells.
Potency Levels:
- Totipotent: Capable of producing all cells of the embryo and extraembryonic tissues (first 4 to 8 cells of an embryo).
- Pluripotent: Capable of producing all cell types of the embryo proper (e.g., inner cell mass cells).
- Multipotent: Capable of forming numerous types of cells specific for a given tissue (e.g., hematopoietic stem cells).
- Unipotent: Capable of generating only one cell type (e.g., spermatogonia).
Committed cells: Make one or very few types of cells.
Progenitor cells: Can proliferate multiple rounds of divisions but are transient and committed to becoming a particular type of differentiated cell, lacking unlimited self-renewal.

Regulation of Stem Cells: Self-Renewal vs. Differentiation

Stem cell fate (self-renewal or differentiation) is profoundly influenced by the stem cell niche, which is the microenvironment surrounding the stem cell.
Regulatory Mechanisms:
1. Extracellular Mechanisms:
  - Physical mechanisms: Involve structural components and adhesion factors of the extracellular matrix (ECM) and cell-to-cell adhesion.
  - Chemical regulation: Driven by secreted proteins (e.g., paracrine, juxtacrine, endocrine signaling) from surrounding cells, as well as neurotransmitter release.
  - Mechanical force: Physical forces can influence stem cell behavior.
2. Intracellular Mechanisms:
  - Regulation by cytoplasmic determinants: Asymmetric localization of specific molecules within the cytoplasm during division can lead to different fates in daughter cells.
  - Transcriptional regulation: Control of gene expression critical for stemness or differentiation.
  - Epigenetic regulation: Heritable changes in gene expression that do not involve alterations to the underlying DNA sequence.

Embryonic Stem Cells

Formation of ICM and Trophectoderm

Early Embryonic Cleavage: Following fertilization, a single mouse embryo undergoes successive cleavage divisions:
- 2-cell stage
- 4-cell stage
- Early 8-cell stage
- Compacted 8-cell stage: Cells begin to compact, increasing cell-to-cell contacts.
- Morula (16 - 64-cell stage): A solid ball of cells.
- Blastocyst: Formation of a fluid-filled cavity (blastocoel). This stage distinguishes:
  - Inner Cell Mass (ICM): Becomes the embryo proper, containing pluripotent embryonic stem cells (ESCs).
  - Trophectoderm: The outer layer that forms extraembryonic structures like the placenta and chorion.

Mechanisms Promoting Pluripotency of ICM Cells

Critical Transcription Factors:
- Oct4, Nanog, and Sox2: These three genes are essential for maintaining the pluripotency of the ICM. Their expression is lost as the epiblast differentiates.
  - Nanog is named after Tír na nÓg, the Irish mythological land of eternal youth, reflecting its role in maintaining an undifferentiated state.
- Cdx2: Expression of this transcription factor is upregulated in the outer cells of the morula, promoting trophectoderm differentiation and actively repressing epiblast (ICM) development.
Apicobasal Polarity and Asymmetric Division:
- Divisions about the apicobasal axis result in daughter cells having different cellular components (e.g., aPKC, E-Cadherin), which is crucial for fate determination.
Hippo Signaling Pathway: Critical for controlling organ size and plays a vital role in ICM formation.
- Initiation: Begins at the cell membrane with cell-to-cell interactions involving adhesion molecules like E-Cadherin. These molecules interact with the F-actin binding protein angiomotin (amot), which triggers the activation of the Hippo kinase cascade.
- In Trophectoderm Cells: Apically positioned partitioning proteins inhibit Hippo signaling, leading to the upregulation of Cdx2 expression.
- In ICM Cells: Activated Hippo signaling represses the Yap/Taz/TEAD transcriptional complex. This repression prevents Cdx2 upregulation and thereby maintains the expression of Oct4, thus preserving pluripotency.

Adult Stem Cells

Presence in Adult Tissues: Many adult tissues and organs (e.g., germ cells, brain, epidermis, hair follicles, intestinal villi, blood) contain stem cells undergoing continual renewal.
Adult Stem Cell Niche: Each adult stem cell resides in and is controlled by its specific niche, which regulates self-renewal, survival, and differentiation of progeny.
Three populations covered: Neural Stem Cells, Hematopoietic Stem Cells, Mesenchymal Stem Cells.

Adult Neural Stem Cell Niche (V-SVZ and SGZ)

Location: In the adult mammalian brain, neural stem cells (NSCs) are primarily found in two regions:
1. The Ventricular-Subventricular Zone (V-SVZ) of the lateral ventricles.
2. The Subgranular Zone (SGZ) of the hippocampus.
- These are sites of adult neurogenesis, extensively studied in rodents but with comparable regions in humans.
Components of the V-SVZ Niche:
- Ependymal cells (E cells): A layer of neuroglia lining the ventricular wall.
- Neural stem cells (B cells): Subtypes B1 (quiescent/inactive), B2, and B3 (actively proliferating).
  - GFAP+ cells with constant Notch activity are quiescent B1 cells.
  - GFAP+/BLBP+ cells with oscillating Notch/Ascl1 show limited proliferation (B2 cells).
  - EGFR+ cells with oscillating Notch/Ascl1 show fast proliferation (B2/B3 cells).
- Progenitor C cells: Transit-amplifying cells.
- Migrating neuroblast A cells: Immature neurons that migrate away from the niche.
  - DCX+ cells with constant Ascl1 undergo proliferation, migration, and differentiation.

Maintaining the NSC Pool with Cell-to-Cell Interaction

The V-SVZ niche is structured and equipped with signaling systems to prevent B cell loss during neurogenic growth or repair.
1. VCAM1 and Pinwheel Architecture:
  - VCAM1 (a cell adhesion molecule) colocalizes with GFAP in B cells at the pinwheel core (the unique arrangement of B cells with their primary cilia extending into the cerebrospinal fluid).
  - Blocking adhesion via VCAM1 antibodies disrupts this pinwheel organization, indicating its structural importance.
2. Notch Signaling: The Timepiece to Differentiation:
  - High levels of NICD (Notch Intracellular Domain) activity support stem cell quiescence.
  - Decreasing levels of Notch pathway activity promote progenitor proliferation and maturation towards neural fates.
  - Notch-Delta signaling and its downstream target Hes (Hairy and Enhancer of Split) genes show temporally oscillating patterns:
    - Increasing oscillations of Notch activity versus proneural gene expression (like Ascl1) progressively promote maturation of B cells to transit-amplifying C cells and then into migrating neural progenitors (A cells).
    - Constant Active Notch/Hes is associated with quiescent B1 cells.
    - Slow Oscillating Hes/proneural is linked to limited proliferation in certain B2 cells.
    - Fast Oscillating Hes/proneural is linked to fast proliferation in other B2/B3 cells.
    - Constant proneural gene expression characterizes proliferating, migrating, and differentiating A cells.

Environmental Influences on the NSC Niche

The NSC niche adapts to changes like injury, inflammation, exercise, and circadian rhythms.
1. Neural Activity: Migrating neural precursors secrete GABA. B cells secrete a competitive inhibitor to GABA, which promotes proliferation in the niche.
2. Sonic Hedgehog (Shh) Signaling: A gradient of Shh contributes to patterning the creation of different neuronal cell types from the V-SVZ.
3. Communication with the Vasculature: Blood-borne factors can influence NSC behavior.
  - Studies with heterochronic parabiosis (joining old and young mice) showed that exposure of old mice to young blood increased vasculature and proliferative neural progeny in their brains.
  - GDF11 secreted from blood vessels is identified as a factor that induces proliferation and neurogenesis.

Hematopoietic Stem Cells (HSCs)

Hematopoiesis: The continuous process of blood cell generation, replacing over 100 billion cells daily.
HSC Function: HSCs divide to produce more stem cells and progenitor cells that differentiate into about a dozen mature blood cell types.
HSC Niche: Located in the bone marrow, divided into two subniches:
1. Endosteal Niche: HSCs adhered to osteoblasts (bone-forming cells) are typically long-term HSCs in a quiescent state.
2. Perivascular Niche: Short-term active HSCs are associated with blood vessels in oxygen-rich areas.
- c-Kit receptor: A marker for HSCs and progenitors, seen in direct contact with sinusoidal microvasculature.
Regulatory Mechanisms in HSC Niches:
- Adhesion to osteoblasts: Keeps HSCs quiescent in the endosteal niche.
- CXCL12 signals: Increased exposure to CXCL12 from CAR cells and mesenchymal stem cells promotes HSC transition into proliferative behavior.
- Downregulation of CXCL12: In the perivascular niche, reduced CXCL12 levels encourage migration of short-term active HSCs into oxygen-rich blood vessels.

Mesenchymal Stem Cells (MSCs)

Unique Plasticity: Unlike most adult stem cells, MSCs demonstrate a surprisingly large degree of plasticity, capable of forming many cell types.
Tissue Distribution: Found in various connective tissues, muscle, eye, teeth, bone, and more.
Dual Roles: Serve as supportive stromal cells within niches (e.g., in the HSC niche) and as multipotent stem cells themselves.
In vitro Properties: A single MSC in culture can self-renew to produce a clonal population that can differentiate to form organs in vitro containing diverse cell types.
- Mesenspheres: MSCs cultured in vitro form 3-dimensional aggregates called mesenspheres, which can produce different cell types like osteoblasts (bone-forming) and adipocytes (fat-forming).
Influence of Substrate Elasticity: MSC differentiation is significantly influenced by the mechanical properties of their environment.
- Cells on soft substrates (e.g., 1 kPa) differentiate into neuronal cells (enhanced eta3 tubulin expression).
- Cells on intermediate stiffness substrates (e.g., 10 kPa) differentiate into muscular cells (enhanced MyoD expression).
- Cells on stiff substrates (e.g., 100 kPa) differentiate into bone cells (enhanced CBF\alpha1 expression).
- Blocking cytoskeleton activity with Blebbistatin inhibits this elasticity-dependent differentiation.

Pluripotent Stem Cells in the Lab

Embryonic Stem Cells (ESCs)

Source: Derived from the Inner Cell Mass (ICM) of the early embryo.
Maintenance in Culture: Their pluripotency is maintained by the same core transcription factors essential in vivo: Oct4, Sox2, and Nanog.
Directed Differentiation: ESCs can be induced to differentiate into various cell types and tissues by exposure to specific combinations of factors and physical constraints.
- For example, controlling factors like Activin, BMP4, Wnt, FGF, VEGF, SHH, Retinoic acid can direct differentiation towards ectoderm (skin, neural), mesoderm (hematopoietic, vascular, cardiovascular, bone, muscle), and endoderm (hepatocyte, pancreatic) lineages.
- Geometric and size constraints of the growth landscape alone can induce significant patterning and differential gene expression, similar to early embryonic development.

ESCs and Regenerative Medicine

Therapeutic Potential: ESCs hold promise for treating diseases (e.g., Alzheimer's, Parkinson's, diabetes) and repairing injuries.
Examples of Preclinical Success:
- Human ESCs cured motor neuron injuries in adult rats by differentiating into new neurons and by producing paracrine factors (BDNF and TGF-\alpha) that prevented existing neuron death (Kerr et al., 2003).
- Precursor cells for dopamine-secreting neurons derived from ESCs completed differentiation and cured a Parkinson-like condition in mice, rats, and monkeys (Kriks et al., 2011).

Induced Pluripotent Stem Cells (iPS cells)

Breakthrough Discovery: Shinya Yamanaka (Nobel Prize in Medicine 2012) and his team reprogrammed somatic cells into ESC-like cells.
Method: Culture of differentiated mouse fibroblasts (e.g., tail tip fibroblasts) infected with viruses carrying pioneer regulatory transcription factors (Yamanaka factors: Oct3/4, c-Myc, Sox2, Klf4). Selection for antibiotic resistance isolates only virus-infected cells, resulting in iPSCs.
Significance: This technology allows for the creation of patient-specific pluripotent stem cells without the ethical concerns associated with embryonic destruction.
Differentiation: iPSCs can generate cells from many lineages, including mesoderm (e.g., red blood cells), ectoderm (e.g., neurons), and endoderm (e.g., pancreatic endocrine cells).
Therapeutic Strategy (Example for Sickle Cell Anemia):
1. Harvest tail tip fibroblasts from a humanized sickle cell anemia mouse model (HbS/HbS).
2. Infect fibroblasts with viruses carrying Oct4, Sox2, Klf4, and c-Myc to generate iPS clones.
3. Correct the sickle-cell mutation (HbS gene) in the iPS cells using specific gene targeting techniques (e.g., CRISPR/Cas9) to produce HbA/HbS iPS clones.
4. Differentiate these corrected iPS cells into embryoid bodies and then into hematopoietic progenitors.
5. Transplant the corrected hematopoietic progenitors back into irradiated mice, theoretically curing the disease.

Organoids: Mini-Organs in a Dish

Definition: Rudimentary 3-dimensional organs cultured in vitro from pluripotent stem cells (ESCs, iPSCs) or adult stem cells (AdSCs).
Purpose: To mimic embryonic organogenesis and to study human organogenesis and patient-specific disease progression at the tissue level in vitro.
Examples: Optic cup of the eye, mini-guts, kidney tissues, liver buds, brain regions (cerebral organoids).
Derivation: Involves specific growth factors, ECM embedding, and controlled culture conditions to promote lineage commitment and self-organization of cells through differential adhesion.
Mini-Gut Example: Intestinal organoids can be cultured to form structures with characteristic crypt-villus architecture and intestinal stem cell niches.
Cerebral Organoids Example:
- Can be derived from human PSCs (hPSCs) by sequential induction (hES media, neural induction media, differentiation media) in 3-dimensional suspension and bioreactor systems.
- These form complex 3-dimensional structures containing neural progenitors (Sox2+) and neurons (Tuj1+).
Disease Modeling: Cerebral organoids are used to model neurological disorders.
- Modeling Microcephaly: Cerebral organoids derived from patients with microcephaly (e.g., caused by Zika virus or genetic mutations like CDK5RAP2) show smaller developed tissues compared to controls.
- Studies revealed that a CDK5RAP2 mutation, involved in mitotic spindle function, leads to low levels of symmetrical division in these organoids, impacting brain size. Providing patient-specific organoids allows for studying disease mechanisms and testing potential therapies.

Summary of Key Concepts

Stem cells self-renew and differentiate into specialized cell types, with potency levels ranging from totipotent to unipotent.
Stem cell behavior is regulated by their microenvironment, the stem cell niche, through extracellular (physical, chemical) and intracellular (cytoplasmic determinants, transcriptional, epigenetic) mechanisms.
ICM pluripotency is maintained by transcription factors Oct4, Nanog, and Sox2, and mechanisms like Hippo signaling, which represses Cdx2 in ICM cells.
Adult neural stem cells reside in niches like the V-SVZ, characterized by a pinwheel organization of B cells.
Notch signaling oscillation dictates neural stem cell quiescent, proliferative, and differentiative states in the V-SVZ.
External factors such as neural activity, Shh gradients, and blood-borne factors (e.g., GDF11) also influence neural stem cell behavior.
Hematopoietic stem cells are regulated by endosteal (quiescence through osteoblast adhesion) and perivascular niches (proliferation and migration influenced by CXCL12).
Mesenchymal stem cells are multipotent adult stem cells found across tissues, acting as stromal support and stem cells, with differentiation influenced by matrix elasticity.
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent stem cells that can be maintained in culture and directed to differentiate into any cell type, offering significant potential for regenerative medicine.
Organoids, 3-dimensional mini-organs from stem cells, are valuable tools for studying organogenesis and patient-specific disease progression in vitro.

Next Steps

Paper Discussion 2